The present invention is directed to methods for synthesizing oligomeric compounds covalently linked to peptides via a linking moiety. The method has particular advantages for larger scale synthesis of peptide linked oligomeric compounds. The present method significantly reduces the cost of preparing peptide linked oligomeric compounds. More specific objectives and advantages of the invention will hereinafter be made clear or become apparent to those skilled in the art during the course of explanation of preferred embodiments of the invention.
Modified oligonucleotides are of great value in molecular biological research and in applications such as anti-viral therapy. Modified oligonucleotides which can block RNA translation, and are nuclease resistant, are useful as antisense reagents. In addition to oligonucleotides that have phosphodiester internucleotide linkages, sulfurized oligonucleotides which contain, for example, phosphorothioate linkages are also of interest in these areas. Because of their chirality (Rp and Sp) phosphorothioate containing oligonucleotides are useful in determining stereochemical pathways of certain enzymes which recognize nucleic acids.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other function, contribute in major proportion to many diseases and regulatory functions in animals and humans. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomers to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The chemical literature discloses numerous protocols for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most routinely used protocols is the phosphoramidite protocol (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein; and The synthesis of Modified Oligonucleotides by the Phosphoramidite Approach and their applications, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage with a suitable reagent effects conversion of a PIII internucleoside linkage to a PV internucleoside linkage. For the purpose of this application, such reagents include oxygen transfer reagents and sulfur transfer reagents. Subsequent hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
Phosphoramidites are commercially available from a variety of commercial sources (included are: Glen Research, Sterling, Va.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; Cruachem Inc., Aston, Pa.; Chemgenes Corporation, Waltham, Mass.; Proligo LLC, Boulder, Colo.; PE Biosystems, Foster City Calif.; Beckman Coulter Inc., Fullerton, Calif.).
Peptide conjugates of antisense compounds have been prepared to enhance the overall effect of these compounds. In order to change pharmacokinetic distribution, cationic groups such as polylysine, polyornithine, polyhistidine and polyarginine and hydrophobic groups such as aromatic aminoacid containing peptides have been covalently linked to oligonucleotides. Peptide ligands targeting cellular receptors have been conjugated to oligonucleotides to enhance cellular permeation. Oligonucleotides have also been modified with peptides that are believed to function as synthetic nucleases.
In one particular study, the thermodynamic melting of a library containing peptide linked oligonucleotides was analyzed to explore the influence of various peptide side chains on the hybridization properties of the DNA (see: Frier, et al., Nucleosides Nucleotides, 1999, 18, 1477-1478; and Harrison, et al., Nucleic Acids Res., 1998, 26, 3136-3145). An invariant 8-mer oligonucleotide was coupled to a five-residue variable peptide region composed of the cationic amino acids lysine, ornithine, histidine and arginine, the hydrophobic amino acid tryptophan, and alanine as a spacer. Melting temperature analysis indicated nearly 1xc2x0 C. increase for each cationic residue present and Tm depended principally on the number of cationic residues. Thus the free energies of binding for polycationic peptide linked oligonucleotides were significantly enhanced compared with the unmodified 8-mer. The origin of this stabilizing effect was derived from a more exothermic enthalpic term. A study of pH dependence showed that the polyhistidine conjugate was particularly sensitive to pH changes near neutrality, as indicated by a significant rise in Tm from 19.5xc2x0 C. at pH 8.0 to 28.5xc2x0 C. at pH 6.0.
In another study, the hybridization properties of a series of oligomers, based on two different 9-mer oligodeoxynucleotide sequences with an appended oligoarginine chain (Rn) were investigated (see: Wei, et al., Nucleic Acids Res., 1996, 24, 55-61). The oligomers were either peptide linked oligonucleotides or peptide-bridged oligonucleotide pairs (e.g., Rn-oligonucleotide or oligonucleotide-Rn-oligonucleotide). For the double-linked probes, it was found that the peptide bridge induces the two 9-mers to bind complementary single-stranded DNA or RNA targets with substantially enhanced thermal stability. Single or double-linked labeled oligomers complexed to complementary RNA were able to activate RNase H.
A synthetic 12-mer oligodeoxyribonucleotide has been coupled at its 5xe2x80x2 terminus to a series of positively charged (xcex4-ornithine)n-cysteine peptides (see: Zhu, et al., Antisense Res. Dev., 1993, 3, 265-275). Site-directed cleavage with RNase H demonstrated that the peptide-modified oligomer hybridized with its RNA target sequence. Increased affinity for target mRNA was also observed.
Melting studies of the complex between an Ha-ras antisense oligonucleotide carrying nuclear localization peptide sequences (NLS) and target mRNA showed that the conjugated oligonucleotide formed a more stable duplex compared with unmodified oligonucleotides (see: Garcia de la Torre, et al., Bioconjugate Chem., 1999, 10, 1005-1012). Despite the presence of the linked peptide, good mismatch discrimination was maintained when the conjugated oligonucleotide was bound to target RNA.
Fusogenic peptides belong to another family of peptides that has been studied in antisense applications. One such fusogenic peptide, derived from the influenza hemaglutinin envelope protein, has been conjugated to antisense oligonucleotides (see: Bongartz, et al., Nucleic Acids Res., 1994, 22, 4681-8468). This peptide changes conformation at acidic pH and destabilizes the endosomal membrane resulting in an increased cytoplasmic delivery of the antisense oligonucleotide. The use of similar fusogenic peptides conjugated to an anti-TAT antisense oligodeoxynucleotide via a disulfide or thioether bond resulted in 5- to 10-fold improvement of the anti-HIV activity of the phosphodiester antisense oligonucleotide on de novo infected CEM-SS lymphocytes in serum-free media. No toxicities were observed at the effective doses (0.1-1 xcexcM). However, no sequence specificity was observed and the fusogenic peptide possessed some antiviral activities on its own (IC50=6 xcexcM). A phosphorothioate (deoxycytidine)28 (S-dC28) peptide conjugate and a streptavidin-peptide-biotinylated S-dC28 adduct showed activity similar to the unconjugated S-dC28 oligonucleotide (IC50: 0.1-1 nM).
Enhanced cellular uptake of oligonucleotides by EGF-R-mediated endocytosis in epithelial cancer cells (A549 cells) has been demonstrated (see: Deshpande, et al., 1996, Pharm. Res., 13, 57-61). To overcome the problem of endosomal entrapment associated with receptor-mediated delivery, the authors evaluated the effects of two fusogenic peptides, polymyxin B and influenza HA2 peptide, for their capability to promote cytoplasmic delivery of oligonucleotides. A conjugate consisting of EGF and poly-L-lysine (PL) was synthesized and complexed with 5xe2x80x2 fluorescently-labeled oligonucleotide. Cellular uptake of this complex in the presence or absence of the fusogenic peptides was monitored fluorometrically and intracellular distribution of the oligonucleotide was determined. Cells treated with the complex exhibited significantly enhanced intracellular fluorescence over controls treated with oligonucleotide alone. Microscopic fluorescence studies revealed, however, that the complex accumulated in endocytic vesicles. Exposure of the cells to the complex in the presence of HA2 peptide and polymyxin B resulted in a more diffused intracellular fluorescence pattern and an increase in fluorescence intensity. These results are consistent with the known fusion and destabilizing activities of the peptides. The uptake of the complex was shown to occur via the EGF receptor-mediated pathway. Since EGF receptors are overexpressed in many cancer cell types, the EGF-PL conjugate may potentially be used as an effective and selective delivery system to enhance uptake of oligonucleotides into cancer cells.
Another approach to the intracellular delivery of oligonucleotides is based on the use of several types of xe2x80x9cdelivery peptidesxe2x80x9d that seem to have the ability to carry large, polar molecules including peptides, oligonucleotides, and even proteins across cell membranes (see: Schwarze, et al., Trends Cell Biol., 2000, 10, 290-295; and Schwarze, et al., Science (Washington, D.C.), 1999, 285, 1569-1572). Two examples of delivery peptides are a 35-amino-acid sequence (xe2x80x9cTatxe2x80x9d) from the HIV Tat protein, and a 16-amino-acid sequence (xe2x80x9cAntxe2x80x9d) from the Drosophila Antennapedia protein. Antennapedia-type peptides have been used to deliver oligonucleotides, including PNAs, into neuronal cells, but their general applicability is yet to be completely studied. Other types of peptides, containing hydrophobic motifs and special recognition motifs, have also been used for antisense delivery.
Ant and Tat peptide-oligonucleotide conjugates have been prepared for the MDR-1 system (see: Astriab-Fisher, et al., Biochem. Pharmacol., 2000, 60, 83-90). The phosphorothioate oligonucleotide component of the conjugates was complementary to a site flanking the AUG of the message for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Both types of peptide-antisense oligonucleotide conjugates, but not mismatched control conjugates, provided substantial inhibition (34%) of cell-surface expression of P-glycoprotein at submicromolar concentrations. The peptide-oligonucleotide conjugates were more potent in the presence of serum than when used under serum-free conditions which is in contrast to cationic lipid-based approaches for intracellular delivery of nucleic acids. Flow cytometry profiles indicated the conjugates accumulated in cells to a much greater degree than the free oligonucleotides. The conjugates reached the nucleus while the free oligonucleotides had virtually no intracellular fluorescence.
Nuclear delivery of antisense oligodeoxynucleotides and selective inhibition of cholesteryl ester transfer protein (CETP) expression by an antisense oligonucleotide complexed to N,N-dipalmitylglycyl-apo E peptide has been shown in a Chinese hamster ovary (CHO) cell line. The cells were stably tranfected with human CETP (Liu, et al., Arterioscler. Throm. Vasc. Biol., 1999, 19, 2207-2213). N,N-Dipalmitylglycyl-apolipoprotein E (129-169) peptide (dpGapoE) has been shown to be an efficient gene delivery system for both plasmids and antisense oligodeoxynucleotides. dpGapoE contains the minimum determinants for binding to both lipid surfaces and the LDL receptor. Thus, dpGapoE could be used to target oligonucleotides to liver. After transfection of oligodeoxynucleotides by dpGapoE, translocation of oligonucleotide to the nuclei and concentration in nuclear structures was observed in  greater than 95% of the cells at 6 and 12 hours by fluorescence microscopy with oligonucleotide observed for  greater than 48 hours. No membrane disruption was observed after transfection. Cellular CETP mRNA levels gradually declined, and the maximum reduction in the mRNA level ( greater than 50%) was observed at 36 hours, after which the mRNA level started to recover. CETP activity in the culture medium declined over 72 hours, with maximum reduction observed at 36 hours (54% of control). Neither CETP mRNA levels nor CETP activity changed after the transfection of sense phosphorothioate oligodeoxynucleotides delivered by dpGapoE complex or naked antisense oligodeoxynucleotides. This is the first demonstration of the use of an LDL receptor-binding peptide for the delivery of antisense oligonucleotides. This approach may enable gene regulation in vivo and development of antiatherosclerotic agents to alter high-density lipoprotein metabolism.
Eighteen conjugates of phosphorothioate oligonucleotides to membrane translocation and nuclear localization peptides were prepared in good yield and were thoroughly characterized with electrospray ionization mass spectra (see: Antopolsky, et al., Bioconjugate Chem., 1999, 10, 598-606). When applied to cells, conjugates exhibiting membrane translocation and nuclear localization properties displayed efficient intracellular penetration but failed to show improved antisense effects. Studies on the intracellular distribution of the fluorescein-labeled conjugates revealed that the conjugates were trapped in endosomes.
It has been demonstrated that conjugates of transporter peptides to PNA show improved delivery and are able to regulate galanin receptor levels and modify pain transmission in vivo (see: International Patent Application PCT/US99/05302, filed Jul. 16, 1999; Pooga, et al., Nat. Biotechnol., 1998, 16, 857-861; and Villa, et al., FEBS Lett., 2000, 473, 241-248). A PNA antisense 21-mer to the human type 1 galanin receptor was linked via a labile cysteine disulfide bond to biotin-labeled peptides known to impart cell membrane permeant properties. These peptides were transportan (galanin (1-12)-Lys-mastoparan (1-14)amide) and pAntennapedia (pAntp (43-58), the third helix of Atennapedia homeodomain). The resulting conjugates improved internalization and down-regulated the human galanin receptor in Bowes cell line and in rat spinal cord in vivo. The intrathecal administration of the peptide-PNA construct resulted in a decrease in galanin binding in the dorsal horn. Due to decreased binding, galanin could not inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs acted in vivo to suppress expression of functional galanin receptors. These peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by an energy-independent pathway (see: Lindgren, et al., Trends Pharmacol. Sci., 2000, 21, 99-103.)
Nine different peptides containing a hydrophobic motif associated with a nuclear localization signal (Chaloin, L., Vidal, P. Lory, P. Mery, J. Lautredou, N. Divita, G., and Heitz, F. Design of carrier peptide-oligonucleotide conjugates with rapid membrane translocation and nuclear localization properties derived from SV40 antigen T separated by various linkers have been synthesized on solid phase (see: Chaloin, et al., Biochem. Biophys. Res. Commun., 1998, 243, 601-608.) The hydrophobic sequence corresponded either to a signal peptide sequence of Caiman crocodylus or to a fragment of the fusion peptide of gp41N, while the hydrophilic sequence was that of a nuclear localization signal. The C-termini of these peptides bear a cysteamide group linked to a fluorescent probe to allow the cellular localization to be determined. The peptide conjugate was successfully synthesized using a disulfide bridge and then used to target fluorescently tagged phosphorothioate oligodeoxynucleotides into fibroblasts. The presence of a linker appears to play a role in the cellular localization. In a 5 minute incubation time more than 90% cells were targeted. It appeared that the membrane-associated conformational state of the peptides was crucial for the internalization process and endocytosis can be ruled out since no temperature (4 or 37xc2x0 C.) effect on the internalization was observed.
The signal peptide (SEQ ID No. 11) should be able to convey oligonucleotides to the endoplasmic reticulum and from there to the cytosol and the nucleus where their targets are located (see: Arar, et al., Bioconjugate Chem., 1995, 6, 573-577.) A 5xe2x80x2,3xe2x80x2-modified pentacosanucleotide, complementary to the translation initiation region of the gag mRNA of HIV, was coupled to a (bromoacetyl)dodecapeptide containing a KDEL signal sequence. The anti-HIV activity of the pentacosanucleotide was compared with that of pentacosanucleotide-dodecapeptide conjugates linked through either a thioether bond or a disulfide bridge. The conjugate with a thioether bond was shown to have a higher antiviral activity than the peptide-free oligonucleotide or the conjugate linked via a disulfide bond.
In another approach, an oligonucleotide-Tat peptide conjugate, having dual binding capability for a designated RNA, was designed (see: Tung, et al., Bioconjugate Chemistry 1995, 6, 292-295.) The peptide portion of the conjugate interacts with a folded domain in the RNA, whereas the oligonucleotide portion hybridizes with a nearby single-stranded region in the RNA. The dual specificity was proven in a model HIV-1 TAR RNA system using an RNase H cleavage assay to assess antisense binding to this RNA. The peptide portion of the conjugate was shown to confer increased specificity on the oligonucleotide.
Antisense oligonucleotides, targeting human immuno-deficiency virus type 1 (HIV), have been linked to fusion peptides derived from the HIV transmembrane glycoprotein gp41 (see: Soukchareun, et al., Bioconjugate Chem., 1995, 6, 43-53.) Thermal denaturation studies showed that the interaction of the conjugate with its complementary strand was similar to that of unmodified oligonucleotides. Thus in this example, the peptide does not confer additional stability to the oligonucleotide-mRNA complex.
A number of methods have been used to synthesize and purify oligonucleotide-peptide conjugates. One method has been developed to fragment couple pre-synthesized peptides to the 2xe2x80x2-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support (see: Zubin, et al., FEBS Lett., 1999, 456, 59-62.) Synthesis of nucleopeptide-oligonucleotide conjugates has been carried out on xcex4-ornithine peptides by modification of the xcex1-amino ornithine functional group with pyrimidyl-1- and purinyl-9-acetic acids or with pyrimidyl-1-and purinyl-9-alanines (see: Sumbatyan, et al., Nucleosides Nucleotides, 1999, 18, 1489-1490.) Nucleopeptides have also been prepared on a solid polymer bearing a photo-activatable linker. Conjugates with the 16-mer oligonucleotide complementary to the env AUG codon region of the Friend murine leukemia virus were prepared in this manner.
Solid-phase synthesis of several peptide-oligonucleotide conjugates has been achieved using a peptide-fragment coupling strategy on a controlled pore glass support (see: Peyrottes, et al., Tetrahedron, 1998, 54, 12513-12522; Peyrottes, et al., Nucleosides Nucleotides, 1999, 18, 1443-1448.) The conjugates contained either a hydrophobic tetrapeptide (Leu-Gly-Ile-Gly) (SEQ ID NO: 19) or an 8-residue basic peptide of the HIV-1 Tat protein coupled to one of two oligodeoxyribonucleotides (an oligoribonucleotide or a mixed ribo/2xe2x80x2-O-Me oligonucleotide). Improved yields were obtained when intemucleotide xcex2-cyanoethyl groups were removed from the support-bound oligonucleotide prior to peptide-fragment coupling, and by use of a long alkyl spacer in the linkage between peptide and oligonucleotide.
Another study describes synthesis of DNA-peptide conjugate molecules on oxime resin (see: Fujii, et al., Pept. Sci., 1999, 35, 293-296.) The oligonucleotide and peptide are covalently linked by cleaving the DNA fragment synthesized on modified oxime resin in the presence of independently prepared peptide fragment bearing free terminal xcex1-amino group and protected side chain residues. This method affords DNA conjugate molecules in moderate to good yields. A different solid-phase synthesis of oligonucleotides conjugated at the 3xe2x80x2termini to a peptide has been developed (see: De Napoli, et al., Bioorg. Med. Chem., 1999, 7, 395-400). A 17-mer antisense oligonucleotide against HIV-1, linked at the 3xe2x80x2-terminus to the tripeptide Gly-Gly-His, was prepared in good yields and characterized by MALDI-TOF mass spectrometry.
A highly basic peptide (net charge +8) derived from the HIV-1 Tat protein was conjugated with quantitative yield to a 19-mer rhodamine-labeled phosphodiester oligonucleotide activated by the pyridinesulfenyl group (see: Vives, et al., Tetrahedron Lett., 1997, 38, 1183-1186.) To avoid precipitation due to antagonist charges of the oligonucleotide and the peptide, the conjugation was performed in high salt concentration (400 mM) and acetonitrile (40%).
Synthesis and characterization of very short peptide-oligonucleotide conjugates and stepwise solid-phase synthesis of peptide-oligonucleotide conjugates on new solid supports have been described (see: Bongardt, et al., Innovation Perspect. Solid Phase Synth. Comb. Libr., Collect. Pap., Int. Symp., 5th, 1999, 267-270; Antopolsky, et al., Helv. Chim. Acta, 1999, 82, 2130-2140). These supports are designed to link the 3xe2x80x2-terminus of an oligonucleotide to the C-terminal end of a peptide via a phosphodiester or phosphorothioate bond in the process of stepwise solid-phase assembly.
Oligonucleotide-peptide complexes offer another non-RNase H mechanism of sequence-specific, hydrolytic cleavage of mRNA. Oligonucleoitde-peptide conjugates designed for mRNA cleavage have been obtained using several methods. By appending a maleimide group to an oligonucleotide, selective coupling to the thiol side chain of a cysteine residue in a peptide has been performed in 53% overall yield (see: Tung, et al., Bioconjugate Chem., 1991, 2, 464-5). Two oligonucleotide conjugates with peptide moieties that either mimic the active site of RNase A (HGH motif) or that contain a Cu(II) complexing metallopeptide (GGH motif) have been synthesized by solid phase synthesis methods with pentafluorophenyl active esters of amino acids and Boc-His(Tos)-OH (see: Truffert, et al., Tetrahedron, 1996, 52, 3005-16.)
Highly efficient endonucleolytic cleavage of single-stranded RNA by a 30-amino acid zinc-finger peptide has been reported (see: Lima, et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 10010-10015.) The peptide sequence corresponds to a single zinc finger of the human male-associated ZFY protein, a transcription factor belonging to the Cys2His2 family of zinc-finger proteins. Interestingly, RNA cleavage was observed only in the absence of zinc. Coordination with zinc resulted in complete loss of RNase activity. The active structure was found to be a homodimeric form of the peptide. Dimerization occurred through a single intermolecular disulfide between two of the four cystines. The catalytic activity was single-stranded RNA-specific; single-stranded DNA, double-stranded RNA and DNA, and 2xe2x80x2-O-methoxy-modified oligonucleotides were not degraded by the peptide. The peptide specifically cleaved after pyrimidines with a preference for the dinucleotide sequence 5xe2x80x2-pyr-A-3xe2x80x2. The RNA cleavage products consisted of a 3xe2x80x2 phosphate and 5xe2x80x2 hydroxyl. The initial rates of cleavage (V0) observed for the finger peptide were comparable to rates observed for human RNases, and the catalytic rate (Kcat) was comparable to rates observed for the group II intron ribozymes. The pH profile exhibited by the peptide is characteristic of general acid-base catalytic mechanisms observed with other RNases. Different chemical methods have been proposed to conjugate this peptide to antisense oligonucleotides (see: International Patent Application PCT/US99/23273, filed Oct. 6, 1999.)
Design of a synthetic nuclease using a zinc-binding peptide tethered to a rhodium intercalator can hydrolyze DNA (see: Fitzsimons, M. P., and Barton, J. K. Design of a Synthetic Nuclease: DNA Hydrolysis by a Zinc-Binding Peptide Tethered to a Rhodium Intercalator, in J. Am. Chem. Soc., 1997, 119, 3379-3380.) A 16 amino acid peptide, DPDGLGHAAKHEAAAK (SEQ ID NO: 18) which binds stoichiometric zinc ion, has been tethered to the DNA-intercalating metal complex Rh(phi)2bpyxe2x80x2(phi=phenanthrenequinone damine, bpyxe2x80x2=4-butyric acid-4-methyl-2,2xe2x80x2-bipyridine) to construct a synthetic DNase. In this combination of DNA-binding and reactive moieties, the rhodium intercalator delivers the appended peptide for reaction with DNA. In the presence of Zn2xe2x80x2, the Rh(phi)2bpyxe2x80x2-peptide conjugate at xcexcM concentration cleaves supercoiled pBR322 DNA and a 17-base pair oligonucleotide duplex under mild conditions. The rate constant for the cleavage of pBR322 DNA by Rh(phi)2bpyxe2x80x2-peptide at pH 6.0 is 2.5xc2x10.210xe2x88x925 sxe2x88x921. Product analysis of cleaved oligonucleotide fragments shows 3xe2x80x2-hydroxyl termini exclusively. These results demonstrate a stereospecific, hydrolytic DNA cleavage reaction by a synthetic complex. Similar experiments with RNA have not been reported.
Systematic studies of the sequence and the structural requirements for good cell penetration and compartmentalization in a range of cell lines as well as correlation with biological activity have not yet been reported for peptide-oligonucleotide conjugates. This is because such studies have been hampered by the often cumbersome and inefficient methods required for the chemical synthesis of such bioconjugates (see: Stetsenko, et al., J. Org. Chem., 2000, 65, 4900-4908). Current synthetic procedures for making peptide linked oligomeric compounds, especially those derived from the cationic peptides, are problematic due aggregation complications associated with electrostatic interactions. Additionally, current methods require excess peptide reagents which render these syntheses difficult, labor-intensive, and economically unfeasible.
Thus, there is a need in the art for cost-effective and efficient methods for the large scale synthesis of peptide linked oligomeric compounds.
The present invention provides methods useful for the preparation of peptide linked oligomeric compounds. The process comprises the steps of:
(a) providing a support medium derivatized with a compound wherein each compound comprises a protected hydroxyl group;
(b) treating the protected hydroxyl group with a deprotecting reagent effective to deprotect the hydroxyl group;
(c) reacting the deprotected hydroxyl group with a nucleoside having a protected hydroxyl group and an activated phosphorus containing substituent group thereby forming an extended compound;
(d) optionally treating the extended compound with a capping agent to form a capped compound;
(e) optionally repeating steps (b), (c) and (d) to form a further extended compound;
(f) treating the capped compound or the further extended compound with an oxidizing reagent thereby forming an oxidized compound comprising one or more nucleosides;
(g) repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising one nucleoside or optionally repeating steps (b), (c), (d), (e) and (f) for oxidized compounds comprising more than one nucleoside to give a further oxidized compound;
(h) cleaving the oxidized compound or the further oxidized compound from the support medium to give the oligomeric compound comprising a linking moiety.
(i) treating the linking moiety attached to the oligomeric compound with a reagent effective to form a reactive sulfur moiety on the linking moiety; and
(j) reacting the reactive sulfur moiety with a peptide the peptide functionalized with a functional group reactive with the sulfur moiety thereby forming the peptide linked oligomeric compound.
In preferred embodiments the nucleoside is a 2xe2x80x2-, 3xe2x80x2-, or 5xe2x80x2-phosphoramidite or a 2xe2x80x2-, 3xe2x80x2-, or 5xe2x80x2-H-phosphonate.
In another preferred embodiment the activated phosphorus containing substituent group is a phosphoramidite, H-phosphonate, phosphate triester or a chiral auxiliary.
In one embodiment, one of either the reactive sulfur moiety or the functional group is xe2x80x94SH and the other of the reactive sulfur moiety or functional group is a disulfide group.
In a preferred embodiment the derivatized support medium is 3xe2x80x2-thiol-modifier C3 Sxe2x80x94S CPG (DMTxe2x80x94Oxe2x80x94(CH2)3xe2x80x94Sxe2x80x94Sxe2x80x94(CH2)3
Design of a synthetic nuclease using a zinc-binding peptide tethered to a rhodium intercalator can hydrolyze DNA (see: Fitzsimons, M. P., and Barton, J. K. Design of a Synthetic Nuclease: DNA Hydrolysis by a Zinc-Binding Peptide Tethered to a Rhodium Intercalator, in J. Am. Chem. Soc., 1997, 119, 3379-3380.) A 16 amino acid peptide, DPDGLGHAAKHEAAAK (SEQ ID NO: 18) which binds stoichiometric zinc ion, has been tethered to the DNA-intercalating metal complex Rh(phi)2bpyxe2x80x2(phi=phenanthrenequinone damine, bpyxe2x80x2=4-butyric acid-4-methyl-2,2xe2x80x2-bipyridine) to construct a synthetic DNase. In this combination of DNA-binding and reactive moieties, the rhodium intercalator delivers the appended peptide for reaction with DNA. In the presence of Zn2xe2x80x2, the Rh(phi)2bpyxe2x80x2-peptide conjugate at xcexcM concentration cleaves supercoiled pBR322 DNA and a 17-base pair oligonucleotide duplex under mild conditions. The rate constant for the cleavage of pBR322 DNA by Rh(phi)2bpyxe2x80x2-peptide at pH 6.0 is 2.5xc2x10.210xe2x88x925 sxe2x88x921. Product analysis of cleaved oligonucleotide fragments shows 3xe2x80x2-hydroxyl termini exclusively. These results demonstrate a stereospecific, hydrolytic DNA cleavage reaction by a synthetic complex. Similar experiments with RNA have not been reported. xe2x80x94O-succinyl-LCAA-CPG).
In one embodiment, hydroxyl protecting groups are acid labile. Preferred hydroxyl protecting groups are trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). These protecting groups are removed by treatment with weak acid preferably dichloroacetic acid or trichloroacetic acid.
In one embodiment, the capping agent comprises 20% acetic anhydride in acetonitrile mixed with about an equal volume of a solution having 20% N-methylimidazole, 30% pyridine and 50% acetonitrile.
In one embodiment, the cleaving step is performed using aqueous ammonium hydroxide. In another embodiment, the cleaving is performed using a bifunctional compound having an internal disulfide group. A preferred bifunctional compound has the formula H2Nxe2x80x94(CH2)2xe2x80x94Sxe2x80x94Sxe2x80x94(CH2)2xe2x80x94NH2.
In one embodiment, the oligomeric compounds of the invention have from about 5 to about 50 nucleosides, with from about 8 to about 30 nucleosides preferred and from about 15 to about 25 nucleosides more preferred.
In another embodiment, methods are provided for preparing a peptide linked oligomeric compound having one of the formulas: 
wherein
T1 is hydrogen or a hydroxyl protecting group;
each X2 is independently, O or S;
each X1 is, independently, O, Pgxe2x80x94Oxe2x80x94, S, Pgxe2x80x94Sxe2x80x94, C1-C10 straight or branched chain alkyl, CH3(CH2)qxe2x80x94Oxe2x80x94, R2R3Nxe2x80x94 or a group remaining from coupling a chiral auxiliary;
g is from 0 to 10;
Pg is CH3, xe2x80x94CH2CH2CN, xe2x80x94C(CH3) (CH3)xe2x80x94CCl3, xe2x80x94CH2xe2x80x94CCl3, xe2x80x94CH2CHxe2x95x90CH2, CH2CH2SiCH3, 2-yl-ethyl phenylsulfonate, xcex4-cyanobutenyl, cyano p-xylyl, diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a blocking group;
each R2 and R3 is, independently, hydrogen, C1-C10 alkyl, cycloalkyl or aryl;
or optionally, R2 and R3, together with the nitrogen atom to which they are attached form a cyclic moiety;
each Bx is, independently, a heterocyclic base moiety;
each R1 is, independently, H, a blocked hydroxyl group, or a sugar substituent group;
n is from 2 to about 50; and
JJ has one of the formulas; 
wherein * denotes the point of attachment to the peptide;
comprising the steps of:
providing an oligomeric compound of the formula: 
xe2x80x83wherein:
L has one of the formulas: 
reacting said oligomeric compound with a peptide having a xe2x80x94SH functional group thereby forming said peptide linked oligomeric compound.
In some embodiments, peptide linked oligomeric compounds have one of the formulas: 
wherein
T1 is hydrogen or a hydroxyl protecting group;
J is C1-C12 alkyl or xe2x80x94(CH2)mxe2x80x94Gxe2x80x94(CH2)mxe2x80x94;
G is O, S, xe2x80x94NHxe2x80x94C (O)xe2x80x94, xe2x80x94NHxe2x80x94C (O)xe2x80x94NHxe2x80x94, xe2x80x94NHxe2x80x94Oxe2x80x94, or xe2x80x94NHxe2x80x94C(O)xe2x80x94Oxe2x80x94;
m is from 2 to about 12;
each X2 is, independently, O or S;
each X1 is, independently, Pgxe2x80x94Oxe2x80x94, Pgxe2x80x94Sxe2x80x94, C1-C10 straight or branched chain alkyl, CH3(CH2)qxe2x80x94Oxe2x80x94, R2R3Nxe2x80x94 or a group remaining from coupling a chiral auxiliary;
g is from 0 to 10;
Pg is CH3, xe2x80x94CH2CH2CN, xe2x80x94C(CH3) (CH3)xe2x80x94CCl3, xe2x80x94CH2xe2x80x94CCl3, xe2x80x94CH2CHxe2x95x90CH2, CH2CH3SiCH3, 2-yl-ethyl phenylsulfonate, xcex4-cyanobutenyl, cyano p-xylyl, diphenylsilylethyl, 4-nitro-2-yl-ethylbenzene, 2-yl-ethyl-methyl sulfonate, methyl-N-trifluoroacetyl ethyl, acetoxy phenoxy ethyl, or a blocking group;
each R2 and R3 is, independently, hydrogen, C1-C10 alkyl, cycloalkyl or aryl;
or optionally, R2 and R3, together with the nitrogen atom to which they are attached form a cyclic moiety;
each Bx is, independently, a heterocyclic base moiety; and
each R1 is, independently, H, a blocked hydroxyl group, or a sugar substituent group;
n is from 2 to about 50; and
nn is from 2 to about 10.